Mosquito NF-κB-mediated innate immunity exerts arbovirus-specific antiviral effects at multiple stages of the viral life cycle

One third of all emerging infectious diseases are vector-borne, with vector ecology and physiology playing key roles in determining whether viruses can access new vertebrate host species and spread globally. Innate immunity is a known barrier to virus replication in mosquito vectors that influences arboviral vector tropism. We here generated novel CRISPR-Cas9-mediated knockouts of the NF-kB family transcription factor Rel2 in Aedes aegypti-derived Aag2 cells and tested the impact on the replication of a diverse range of arboviruses in the Flaviviridae and Togaviridae families and the class Bunyaviricetes. We found that NF-kB-mediated innate immunity has broad antiviral activity against the Ae. aegypti-borne orthoflaviviruses dengue virus (DENV), yellow fever virus (YFV) and Zika virus (ZIKV) in mosquito cells. In contrast, little impact of NF-kB-loss-of-function was observed for the alphavirus chikungunya virus (CHIKV) or phlebovirus Rift Valley fever virus (RVFV), indicating specificity in the antiviral effects of NF-kB-mediated immunity. By comparing orthoflaviviruses with different transmission routes (mosquito-borne, tick-borne, no known vector), we demonstrated that NF-kB-mediated immunity exerts its antiviral effects both early and late in the viral replication cycle, and that NF-kB-mediated immunity is not the only molecular barrier influencing the ability of orthoflaviviruses to replicate in Ae. aegypti cells. Overall, our work demonstrates the importance of mosquito NF-kB-mediated innate immunity in suppressing arbovirus replication, and shows that the barriers for arboviruses to adapt to new vector species are multifactorial and virus-specific. Our findings increase our understanding of the molecular barriers influencing arboviral emergence, and could inform the development of refractory mosquitoes incapable of transmitting human pathogens.